Four-micrometer-thick tissue sections were mounted on positively charged barrier frame slides, dewaxed in xylenes, and rehydrated through an ethanol dilution series (100% to 25%). Tissue sections were digested with 5 μg/mL of proteinase K for 20 minutes at 37°C to facilitate probe penetration and exposure of miRNA species. To minimize nonspecific binding based on charge interactions, tissues were subjected to a brief acetylation reaction [66 mmol/L HCl, 0.66% acetic anhydride (v/v) and 1.5% triethanolamine (v/v) in RNase-free water]. Then, tissue sections were prehybridized at the hybridization temperature (see Supplementary Table S1 for details) for 30 minutes in prehybridization solution which consisted of 50% deionized formamide, 5× sodium chloride/sodium citrate buffer, 1× Denhardt’s solution, 500 μg/mL of yeast tRNA, and 0.01% Tween. The prehybridization solution was replaced with 200 μL of hybridization solution containing 10 pmol of the hapten-labeled LNA probe and tissues were incubated for 90 minutes at the hybridization temperature and washed thrice for 10 minutes in sodium chloride/sodium citrate buffer at the established stringency of sodium chloride/sodium citrate (see Supplementary Table S1 for details). At this point, tissue slides were loaded onto the Biogenex i6000 staining machine (BioGenex Laboratories, Inc.), which was programmed to dispense 400 μL of the appropriate reagent per step. Slides were treated with 3% H2O2 to inactivate endogenous peroxidase and block with 5% bovine serum albumin in PBS (w/v). Followed by primary and secondary antibody incubation in PBT [1% bovine serum albumin (w/v), 0.1% Tween 20 (v/v) in PBS] and washes in PBST [0.01% Tween 20 (v/v) in PBS], tyramine-conjugated fluorochrome was applied to the slide and the tyramide signal amplification (TSA) reaction was allowed to proceed for 10 to 30 minutes. Sequential TSA rounds for the detection of other miRNAs, noncoding RNAs, or proteins followed the same protocol. Finally, slides were washed extensively with PBST and mounted with antifading ProLong Gold Solution (Invitrogen) with or without 4′,6-diamidino-2-phenylindole (for nuclear counterstaining). Please see Supplementary Table S2 for a list of antibodies used and Supplementary Table S3 for the preparation of tyramide-conjugated fluorescent substrates.